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    Journal of Pharmaccuical and Biometical nabs 49 (2000) 108-114 Contents lists available at ScienceDirect ae] Journal of Pharmaceutical and Biomedical Analysis ELSEVIER journal homepage: www.elsevier.com/locate!jpba Determination of nicotine, cotinine, and related alkaloids in human urine and saliva by automated in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry Hiroyuki Kataoka’, Reiko Inoue, Katsuharu Yagi, Keita Saito School o army Shfisu ives, Nsigawara Okoyma 705-516. fpae ARTICLE INFO ABSTRACT ‘rice ory Receiied 27 August 2008 Receved neve fort 205eprember 2008 ‘Accepted 25 September 2008 Fiabe online #Ocober 2008, | simple, rapid and sensitive methed forthe determination of nicotine, cotinine, 1 anatabine in human urine and saliva was developed, These compounds we intube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS), Nicotine, cotinine and related alkaloids were separated within 7min by high performance Tiguid chromatography (HPLC) using a Synergi du POLAR-RP 80A columa and SmM ammonium for- ‘matejmethanol (55/45, vjy) as a mobile phase at 2 low-rate of 08 ml|min. Electrospray ionization conditions in the positive ion mode were optimized for MS detection ofthese compounds. The optimum | ube SPME conditions were 25 daw ject cycles with a sample sizeof 40 sing a Ch-Pora PLOT fe ine capllay columnas te extraction device. The xtactd compounds cout be desored easily fom Caine the eapilary by passage ofthe mobile phase, ano carryover as observed. Using te in-tube SPME Lguidchomatogapty-mastspedromety LC-MS method, the elation curves were near inthe concentration ange of 05-20 gh nicotine Sioking cotinine and related compounds in urine and salva andthe detection iis (S]N=2) wer 1-0 pgm “The method described here showed 20~s-fld higher sensivty than the dnec injection method (3 injection} The wthin-runand between-day precsion (relative standard deviations) wete below 47% 13% (ne5), respectively. Tis method was applied succesfully to analysis of urine and saliva samples without interference peaks, The recoveries of ncoine, cotinine andvelated compounds spiked into urine In sallvs samples were above #3% and the relative standard deviations were below 7k Ts method trated to any urinary and salivary level of hese compounds in nicotine inate and smoking 12008 Elsevier BY. al rights eseved 1. Introduction racologically active substance in tobacco [5] and is suspected to contribute to human diseases, such as cardiovascular and repro- ‘The use of tobacco products may be most critical public health ductive disorders [6,7]. There is good evidence than most smokers problem [1] Its well known that tobacca smoke is a majar cause of mortality and morbidity (2), More than 4000 compounds have been identified in tobacco smoke, at least 50 of which have been shown to be carcinogenic [3] Epidemiological studies in smokers have indicated a dose-response relationship between the number of cigarettes smoked per day and the risk of developing certain smoking-related diseases [4], Tobacco alkaloids ate the active pri cipal components in all tobacco products. Among more than 20 different alkaloids found in tobacco, nicotine is the most abun- dant (98% of the total alkaloids) and accounts for widespread human use of tobacco products throughout the world because of its addictive properties, Furthermore, nicotine is the major phar- corresponding autor, Tel: +81 86271 #342; fx: +81 86271 242, ‘Pinal ede haaoladshutsuaejp (Kataoka) (731-7085 - sce font matter © 2008 Elsevier BN. Alright eserve 4101016) b.2008. 08.048 are dependent on nicotine and that the severity of tobacco depen- fence may be related to the level of nicotine intake. The minor alkaloids, including nornicotine, anabasine, and anatabine are also pharmacologicaly active. However, they are ess potent than nico- tine [8]. Nicotine is absorbed rapidly in humans through the skin and mucosal lining of the mouth and nose or by inhalation in the lungs, and exerts a number of physiological effects in both active and passive smokers, defined as cigarette smokers and ‘non-smokers exposed to environmental tobacco smoke. It is esti- ‘mated that an average of 70-80% of the nicotine absorbed by a smoker is metabolized to cotinine [9]. Nicotine and its metabo- lite cotinine can be measured in various biological luis, including blood, saliva, and urine |10,11]. Therefore, these compounds have been widely used as biological markers to determine tobacco smoking status and estimate exposure to environmental tobacco smoke [10-14], Serum nicotine and urinary/saliva cotinine have take Journal of Practical and Biri Anas 49 (2000) 108-114 109 also been used to guide the dose of nicotine replacement therapy 115,16} ‘Nicotine, cotinine, and related alkaloids in various biological fluids have been determined by radioimmunoassay (17), enzyme immunoassay [18.19], g25 chromatography (GC) [20,21], GC with mass spectrometry (GC-MS) [20-22]. high performance liquid ‘chromatography (HPLC) [20], and LC with tandem massspectrome- ‘ry(LC-MS-MS) [23-29], The immunological methods are sensitive but have cross-reactivity with nicotine and related compounds, ‘thus leading to overestimation, LC-MS-MS can provide a sensitive and selective means for comprehensive measurement of nicotine {and its metabolites. However, mast of the above methods generally require time-consuming sample preparation procedures, such as liguid-tiquid extraction or solid-phase extraction, to remove coex- isting substances in biological samples prior to analysis except for some LC-MS-MS methods [24.26]. In-tube solid-phase microextraction (SPME) (30), using an open tubular fused:-sitica capillary with an inner surface coating as the 'SPME device is simple and can be coupled easily on-line with HPLC ‘and LC-MS. In-tube SPME allows convenient automation of the extraction process, which not only reduces the analysis time, but also provides better precision and sensitivity than manual off-line techniques. We recently developed an in-tube SPME method for the {determination of urinary drugs 31.32] and salivary cortisol [33] by coupling with LC-MS. The details of the in-tube SPME technique and its applications have also been summarized in a number of reviews [34-36]. Here, we report an automated on-line in-tube ‘SPME LC-MS method for simultaneous determination of nicotine, cotinine, and related alkaloids in urine and saliva samples. These samples can be obtained easily and salivary cotinine level has been reported to be an especially good indicator of plasma cotinine con~ centration [37]. Using this method, we also analyzed the changes in ‘urinary and salivary levels of these compounds in nicotine intake and tobacco smoking. 2. Experimental 24. Materials Nicotine, cotinine, nornicotine, and anabasine were purchased from Sigma-Alcrich Japan (Tokyo, Japan) and anatabine was purchased from Toronto Research Chemicals (North York, ON, Canada). Each compound was dissolved in methanol to make 2 stock solution at a concentration of 1mgimL. Each solution ‘was stored at 4°C and diluted to the required concentrations ‘with pure water prior to use, LC-MS grade methanol and dis tilled water used as mobile phases were purchased from Kanto Chemical (Tokyo, Japan). All other chemicals were of analytical rade, 22. Instrument and anaiytical conditions ‘The LC-MS system was a Model 1100 series LC coupled with, an atmospheric pressure electrospray ionization (ESI) MS (Agi- lent Technologies, Boeblingen, Germany). A Synergi 4u POLAR-RP 804 column (150mm x 4.6mm ia, particle size of 2.5 wm) from Phenomenex (Torrance, CA, USA) was used for LC separation. LC Conditions were as follows: column temperature, 30°C; mobile phase, SmM ammonium formate/methanol (55/45, v/v) and flow- Fate, 08 ml/min (during the in-tube SPME treatment, the flow-rate ‘was set (0 0.2 mL|min to save mobile phase solution), ESI-MS con- ditions were as follows: nebulizer gas, Ny (55psi); drying gas, Nz (12L/min, 350°C); fragmenter voltage, NOV: capillary volt~ age, 2500; ionization mode, positive mode; mass scan range, {60-200 amu; selected ion monitoring (SIM).mjz 149 nornicotine), mz 161 (anatabine), m[z 163 (nicotine and anabasine). and mjz 177 (cotinine) and dwell times for the ions in SIM, 144ms. LC-MS data ‘were processed using an HP Chemstation (Hewlett-Packard, Palo Alto, CA, USA), 23. In-tube solid-phase microextraction ‘ACP-Pora PLOT amine capillary column (60 cm x 0.32 mm id 10m film thickness: Varian inc. Lake Forest, CA, USA) was used 28 the in-tube SPME device. The column was placed between the injection foop and injection needle of the autosampler, and the injection loop was retained in the system to avoid fouling of the metering pump. Capillary connections were facilitated by use of 2.2.5-cm sleeve of 1/16-in polyetheretherketone (PEEK) tubing at ‘each end of the capillary (1 in =254.m). A PEEK tubing (330 pm i.) was found to be suitable to accommodate the capillary used. Normal 1/16-in stainless stee! nuts, ferrules, and connectors were then used to complete the connections. The autosampler software ‘was programmed to control the in-tube SPME extraction, desorp- tion, and injection, Vials (2m) were filled with 1.0 mL of sample for extraction, and set into the autosampler programmed to con- ‘ol the SPME extraction and desorption technique. In addition, 15m aliquots of methanol and water in 2 mL autosampler vials with a septum were set on the autosampler. The capillary col umn was washied and conditioned by two repeated draw/eject cycles (40 Leach) of these solvents, and then a 50-wL air plug ‘was drawn prior fo the extraction step. The extraction of cor- tisol onto the capillary coating was performed by 25 repeated ‘draw/eject cycles of 40 uL of sample ata flow-rate of 150 L/min ‘with the six-port valve in the LOAD position. After washing the tip of the injection needle by one drawjeject cycle of 2yL of methanol, the extracted compounds were desorbed from the cap- ilary coating with mobile phase flow. Then, the compounds were transported to the LC column by switching the six-port valve (0 the INJECT position, and detected by the MS system with SIM mode. During the analysis, the SPME capillary was washed and Conditioned with mobile phase for the next extraction. Outline of the in-tube SPME/LC-MS system is shown in previous papers, (33.36) 24. Sample preparation Urine samples from healthy volunteers were collected in glass bottles and processed immediately or stored at ~20°C until use, Saliva samples were collected in Salisoft tubes containing a polypropylene-polyethylene sponge (Assist, Tokyo, Japan), and the tubes were centrifuged at 2500 xg for Smin to elute the saliva, Urine or saliva solutions (0.1-0.2 mL) were added to 0. mLof 2M acetate bufler (pH 5) and the total volume was made up to LOmL ‘with distilled water, The mixtures were used for the following in-tube SPMELC-MS analysis. Standard mixture was added to con- ‘tol urine and saliva samples (which did not include nicotine or Felated compounds) at concentrations of 0.5, L0, 2.0, 5.0, 10, 20, and 50 ng/ml of each compound, and calibration curves were con- ‘structed, Urinary creatinine concentrations were determined by the Jaffé method using a creatinine test kit (Wako Pure Chemicals, Osaka, japan). 25. Nicotine intake and smoking ‘The aim ofthe experiment was explained to the subjects before hand and consent was obtained after confirmation that they fully understood the experiment. The non-smoking subject consisted ‘of 52 male volunteers who chewed Nicorette® (Pfizer Co, Ltd, no 1. Ktook orl of Pharmacol an Biomedical Analysis 492000) 18-114 ‘Tokyo, Japan) gum containing 1mg of nicotine for 30min from, 8:00 am, Urine and saliva were sampled just before nicotine intake and after 2, 4, 6, 9, 12, 15, and 21h. The smoking sub- ject consisted of 22 male volunteers who smoked a cigarette (including 03mg nicotine) at 9:00 and 15:00 after stopping smoking the day before. Urine was sampled just before smok- ing and after 2. 4, 6, 8, 10, and 12h. Urine samples were also collected from smokers (active smoking) and non-smokers (pas- sive smoking). The collected samples were stored at -20°C until assayed, 3. Results and discussion 3.1 LC-MS analysis of nicotine and related compounds For MS operation, ESI positive ion mode was evaluated for the determination of nicotine and related alkaloids. To select the moni- Coting ion for these compounds, the ESI mass spectra were initially analyzed by LC-MS with direct liquid injection into the column. Nicotine and related compounds gave [M+H]” as a base ion in the mass scan range of 60-200.amu. The [M+H-NH3]" was also observed in nomnicotine (m/z= 132.2) and anatabine (m/2= 1441) Parameters, including nebulizer gas pressure, drying gas flow-rate, lragmenter voltage, and capillary voltage were optimized by flow injection analysis LCseparation of nicotine and related compounds was performed, using a Synergi 4u POLAR-RP SOA column. As shown in Fig. 1, these compounds were eluted within 7 min using SmM ammonium for- ‘mate/methanol (55/45, v/¥) as a mobile phase, at a flow-rate of 0.8 ml/min. Nicotine and related compounds could be detected selectively in SIM mode. ‘won| OTIC sae | CDH Nomctnw eee oon | (C)miz=t64 ne | OPENS — oat eur are ection ‘ow| ©1177 cotine Rearicurece tere arse ern Fig. 1. Chromatograms obtained from 100 ng/mb standard compounds by dret injection. (4) Tol ion chromatogram (B)-(E selected fon chromatograms. See Seton for LMS conditions Peak height count g 3 Fig 2 Eecs ocapilay costings on the insube SPME of nicotine and elated com pounds. ach compound was etracted by 20 drawleect eyes o 40 Lo standard ‘olution Daal of exch) ata rate of 150 Lm, 3.2, Optimization of in-tube solid-phase microextraction and desorption To optimize the extraction of nicotine and related compounds by in-tube SPME, several parameters, such as the stationary phase of the in-tube SPME capillary column and number and volume of draw/eject cycles were investigated. Extraction efficiency in in- tube SPME was evaluated by comparison of peak height in each condition, ix different capillary columns, CP-Sil SCB (Varian Ine, Lake Forest, CA, USA, 100% polydimethylsiloxane, 5 um film thick: ress), CP-Sil 19C8 (Varian, 14% Cyanopropyl phenyl methsiloxane, 1.2 jam film thickness), CP-Wax 52CB (Varian, Polyethylenegly- col, 12m film thickness), and CP-Pora PLOT amine (Varian, basic modified stylene divinytbenzene polymer, 10 um film thick- ness), Carboxen 1006 PLOT (Supelco, Bellefonte, PA, USA, carbon ‘molecularsives, 15 yam fllm thickness) and Supel Q PLOT (Supeleo, Divinylbenzene polymer, 17 um film thickness) were tested as extraction device. In in-tube SPME, the amount of analyte extracted into the stationary phase of capillary column depends on factors such asthe surface area, film thickness and polariy ofthe capillary coatings. As shown in Fig. 2, the extraction efficiency of the porous polymer-type capillary column was better than those ofthe other Columns. As the PLOT column has a large adsorption surface area and thick flm layer, the amount extracted was greater than that With liquid-phase-type columns. Among the PLOT column, a CP- Pora PLOT amine gave superior extraction efficiency because of its affinity to relatively polar compounds. ‘With in-tube SPME, the extraction time, flow-rate, and sample pH are related to the amounts of compounds extracted, To monitor the extraction time profile of nicatineand related compounds by in- {ube SPME, the number of draw ejecteyeles was varied from 0t0 25 using aCP-Pora PLOTamine capillary column. Asshown in Fig. 3(A), the extraction equilibrium of these compounds was not reached with 25 draw/eject cycles of 404L of sample. Although extrac- tion equilibrium is incomplete itis possible to cease extraction before equilibrium to reduce the analysis time, because quantita- tive reproducibility is obtained by fixing SPME conditions using an autosampler. Draw/eject rate in in-tube SPME was tested at 50, 100, 150 and 200 iL/min. Extraction efficient is highest at 50 l/min, and it decreased slowly with increase of drawfeject rate. In this ‘method, a draweject rate of 150 L/min was used as optimal flow- rate. Below this level, extraction requires an inconveniently long ‘ime, and above this level, Bubbles tend to form inside the capil- Jary column, reducing the extraction efficiency. The effect of the Hof the sample matrix on extraction of nicotine and related com- pounds was examined using several buffer solutions. As shown take Journal of Practical and Biri Anas 49 (2000) 108-114 (A) s0om0 m (8) 00000 Fi. 3. eet of (A) drawieet cele and (flaw ate on the in-ube SPME of nicotine and related compounds, Fach compound was extracted by dale of 40. of Standard slation 20g of ach) using CP-Pora PLOT amine capa. in Fig, 3(8), acetate buffer (pH 5 or 6) was more effective, and the optimal concentration of this butfer was 20mM. The abso- lute amounts ofthese compounds extracted by the SPME capillary column were calculated by comparing peak area counts with the corresponding direct injection of the sample solution onto the LC column. At a sample concentration of 20ngimL, 5.1 ng (25.5%) of nicotine, 5.31g (26.5%) of cotinine, 2.4ng (12.0%) of nornicotine, 2.7 ng (13.5%) of anatabine, and 2.6ng (13.0%) of anabasine were ‘extracted onto the CP-Pora PLOT amine column by in-tube SPME. Although the extraction yields of these compounds were rela- Lively low, they showed good reproducibility (RS.D.<5%) due tothe autosampler. ‘The mobile phase was found to be suitable forthe desorption of nicotine and related compounds extracted into the stationary phase (of the capillary column, Dynamic desorption of these compounds from the capillary could be achieved readily by switching the six- port valve of LC-MS instrument. The desorbed the compounds were ‘transported to the LC column by mobile phase ow. Air plugging before the extraction step was carried out to pre- vent not only sample mixing butalso desorption of analyte fromthe capillary coating by the mobile phase duting the ejection step. No carryover was observed because the capillary column was washed and conditioned by drawjeject cycles of methanol and mobile pphase prior to extraction, The extraction and desorption of nico- tine and related compounds by the in-tube SPME method were accomplished automatically within 30 min, and automated analysis, fof about 48 samples per day was possible by overnight opera- tion, 3.3. Sensitivity linearity and precision Nicotine and related compounds provided an excellent response. in ESI-MS. As shown in Table 1, the detection limits ofthese com- pounds were 15-40 pg/mL with signal-to-noise ratios of 3:1 under ‘Our LC-MS conditions. The in-tube SPME method was 20~46-fold more sensitive than the dizect injection method (5 uL injection). because these compounds were concentrated in the capillary col- umn during draw/eject cycles. Sensitivity ofthis method was about 10 times higher than those of LC-MS-MS method reported pre- viously [23-29]. The calibration curves for nicotine and related compounds were constructed from the peak height counts. AS shown in Table 1, a linear relationship was obtained for each com- pound in the range 0.5-20 ng/mL urine (six-point calibration) and the correlation coefficients were above 0.9969. On the other hand, ‘the within-run and between-day precision (relative standard devi ations, RS.D.) tthe concentration of 2ng/mL were below 4.7% and 11.3% (n=5), respectively (Table 1). 34. Applicaton tothe analysis of urine and saliva samples Saliva samples were collected easily using Salisoft tubes con- taining a polypropylene-polyetiylene sponge. Urine and saliva samples could be analyzed directly by the dilution of the sample ‘without any other pretreatment. The recovery rates of nicatine and related compounds added to urine and saliva samples by com- parison with pure standard sample were 30-57% and 43-75%, respectively. The lower recoveries were corrected by using cal bration curves of nicotine and related compounds spiked into the ‘pooled urine and salivaas described in Section 2s shown in Fig. 4, the urine and saliva samples were analyzed successfully without interference peaks by SIM mode detection. To confirm the validity ‘of this method, known amounts of nicotine and related compounds ‘were spiked into 0.1 mL of pooled urine and saliva samples, and their recoveries were calculated, As shown in Table 2, the recaver- ies of these compounds were above 83-98% and relative standard deviations were below 71% Table near egression data, detection limits and withn-ran and between day precios of lctine and elated compounds by in-tubeSPMELC-NS. Compound SIM (le) Rearesson ine oration Detection iit hain DH Within-run—‘Between-day Slope lntecept Coefficient __Dieetinjection ——_UntubesSPME Ratios RSL SDR ‘Anatabine 161 nm” 185, 09975 a5 boat meu 21 ‘mabasine 182 ‘eto 702 0042 se oss Ba ua 2s ‘cotine 13 Tei 1590, 0060 124 ox a3 ua. 208 Cetnine fd 31708 can fan004 O34 ois nr 0s 134 © Callbration ange: 05-20 g/l, spat (a= 18) SIN=3 « Senstvty ate of ret injection method agains in-tube SPME metho, ones, me 1. Ktook orl of Pharmacol an Biomedical Analysis 492000) 18-114 (1) Urine (0.2 mL) (2) Sativa (0.2 mL) 20009] ATIC soano| A Oey re hte eta ees rr a ee er a 2000] (©) metas ooo | (8) m2=148 a ° oT see eT min re) +2000 () mve=t64 sooo | (0) 2-181 en ooo Se sateteds eatery eens ieraamn Sees aot ateo feepeare eres orien zoo] (0) mz=169 eo so | (0) m2=169 cree em ectie nish omen uth mi 20000] €) m2=177 sco | (E) 120177 Oars eterno ratu trea venir eer tel tam | earl ee egestas erear aver Fig. Chromatograms obtained from rine an saliva samples ater ncoine nak, Se Section 2 frin-tbe SPMEILC-MS conditions 35, Excretion of nicotine and related compounds by nicotine intake and smoking Urinary nicotine and cotinine contents are useful biomarkers, to evaluate smoking [10-14]. To evaluate the utility of the devel- ‘oped method, we analyzed the influence of the intake of nicotine. ‘The test involved chewing Nicorette gum containing 1 mg a nico- tine for 30 min from 9:00 am. and urine and saliva were sampled just before nicotine intake and after 2, 4,6, 9, 12,15, and 21. AS shown in Fig. 4, nicotine and cotinine were detected in urine and saliva samples, but the other compounds tested in this study were not detected. As shown in Fig. (A), the urinary nicotine content reached a maximum level after 2h and subsequently decreased by degrees, The urinary cotinine content reached a maximum level alter 4h, On the other hand, the salivary nicotine content increased transiently after 2h, while the cotinine content hardly increased (Fig. 5(B)) To evaluate the urinary excretion of nicotine and cotinine with smoking, subject who was smoker smoked acigarette contain- ing 0.3 mg of nicotine at 9:00 and 15:00 after stopping smoking the day before. As shown in Fig. 6, the urinaty nicotine and coti- pine contents increased with smoking. As shown in Table 3, urinary excretion of nicotine and cotinine also increased depending on the number of cigarettes smoked in one session. Furthermore, urinary excretion of these compounds increased in non-smoker associated with passive smoking, These results suggest that urinary excretion and salivary secretion of nicotine and cotinine sufficiently reflect active and passive smoking of cigarettes, and it was confirmed that these compounds are useful biomarkers to evaluate smoking. table? Recoveries of nicotine and elated compounds spike ino urine and salva samples Compound Spiked (ant) Recovery Gnean 15D (e=3) ie Sie ownage RSD) Nowmiotne 2 wae 61 za 2 os 40 44 Anatabine 2 os oog.228 a 20 2 942225 26 Anabasine 2 35 piven) 29 2 10 9535 45, 47 Nictine 2 7 sos29 as 2 2 952220, 2 Cine 2 oo sage 2 20 19 965120 a take our! of Practica and Birdie Ana 49 (2000) 109-114 Urinary exertion of icetine and coinine native and pase smoking ry aren span an aia Eee =

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